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Abstract: Intrinsically disordered proteins (IDPs) is a term used to describe proteins that do not have a well-defined tertiary structure. IDPs have many roles such as in cell cycle control (p53), neuronal signal transmission (myelin basic protein), and protein stability (dehydrins). Producing recombinant IDPs in bacteria for nuclear magnetic resonance (NMR) studies is problematic because the lack of stable tertiary structure makes them excellent substrates for bacterial proteases, which will cause loss in yield. We have developed a two-step method to produce the grape dehydrin K2 and YSK2 using Escherichia coli. Dehydrins are expressed by certain plants in response to dehydration, increased salinity, or low temperatures. Purification of 10mg/L (K2) and 15mg/L (YSK2) was performed by boiling bacterial pellets to lyse the cells, remove most of the contaminating proteins, and denature bacterial proteases. This resulted in protein purity comparable to that produced by sonication and nickel affinity chromatography. Boiling was followed by cation exchange chromatography to remove the remaining trace contaminants. The sample was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography. The method presented here can easily be adapted to the purification of other IDPs and heat-stable proteins without requiring multiple chromatography steps or the use of protease inhibitors. [Copyright &y& Elsevier]

Summary: A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28kDa, pH and temperature optima for this protease were 10 and 60°C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu2+ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents. [Copyright &y& Elsevier]

Abstract: A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5mg of purified protein was obtained from 1.0g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC50 was about 58nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC50: 100nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP. [Copyright &y& Elsevier]

Abstract: Invertase is a major protein of grape juice and wine. Accordingly, in order to study the biochemical and structural characteristics of this protein and for a better understanding of its physico-chemical properties, large amounts of the pure protein are needed. A simple method for the purification of the grape vacuolar invertase in a preparative-scale is described in this work. The grape protein was isolated and purified from must by ultrafiltration and anion exchange chromatography. The identification and purity determination of the grape invertase fraction were assessed by SDS-PAGE, and were then confirmed using nanoLC-chip-MS/MS analysis. The laboratory fractionation procedure presented in this work generated large quantities of pure grape vacuolar invertase from must. [Copyright &y& Elsevier]

Abstract: A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25mM MOPS, 1.5M NaCl, 0.1mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis. [Copyright &y& Elsevier]

Abstract: An IgM antibody was purified from hybridoma supernatant containing serum using a three-step purification process comprising of tangential flow filtration, anion-exchange chromatography and size-exclusion chromatography. Recovery and purity were significantly improved upon adaptation of the hybridoma to serum-free media. The process could even be simplified by omitting the initial tangential flow filtration step. Even with a two-step purification process a purity of >98% and a recovery of >60% was obtained. [Copyright &y& Elsevier]

Abstract: The present work deals with the purification and drying of bromelain, one of the vegetal proteases. The purification of the enzyme from fruit Ananas comosus was carried out by precipitation and by chromatography. Further, the drying of chromatographically purified fraction was carried out in freeze dryer and spray dryer. The activity was expressed in terms of casein digesting units (CDU). The bromelain extract of higher purity was obtained from the fruit using ion exchange chromatography. The elution efficiency in chromatographic purification was obtained in the range of 80–90%. The fold purity obtained by chromatography was 3.3 times as compared to that obtained by precipitation. Bromelain thus obtained was subjected to spray and freeze drying. It was observed that the yield obtained in spray dryer at an outlet temperature of 40–50°C was almost 50–70% and in case of freeze dryer the yield obtained was 96%. The resulting residual activity and the specific activity was compared between spray-dried and freeze-dried powder. The purity of enzyme obtained in the present study was found to be 2.8 times more than that of a commercial sample. [Copyright &y& Elsevier]

Papain, a proteolytic enzyme was purified from latex of Carica papaya by a variety of techniques such as two-step salt precipitation, organic solvent precipitation (ethanol) and ion exchange chromatography. Fold purity obtained using ion exchange chromatography was in the range of 4.5 to 5 times as compared to the precipitation techniques. The purified papain was entrapped in ionotropically cross-linked alginate beads, in order to develop formulation for site-specific delivery of papain in intestine. The entrapment efficiency for papain in alginate beads was found to be in excess of 86%. Drying at low temperature and low air velocity results into maximum retention of papain activity; however, drying time increases significantly. The dissolution study shows the possible use of alginate beads for site-specific intestinal delivery of papain. The shelf-life of papain can be improved significantly by entrapment in alginate matrix, which was confirmed by an FTIR study. [ABSTRACT FROM AUTHOR]

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43kDa. It was optimally active at 50°C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45°C for 90min. K m value for polygalacturonic acid was 1.63mg/mL and the corresponding V max was 677.8μM min−1 mg−1. The inhibition constant (K i) for gluconic acid d-lactone was 20.75mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site. [Copyright &y& Elsevier]

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH4)2SO4, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7U/ mg protein) was a monomeric protein with a molecular mass of 35.0kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45°C and 5.0, respectively. The xylanase was activated by Cu2+ up to 115.8% of activity, and was strongly inhibited by Hg2+, Pb2+ up to 52.8% and 89%, respectively. The xylanase exhibited Km and V max values of 3.267mg/mL, 18.38M/min/mg for beechwood xylan and 3.747mg/mL, 11.1M/min/mg for birchwood xylan, respectively. [Copyright &y& Elsevier]

A simple single step ion exchange chromatography method has been developed for purification of SA from plant cell culture extracts. The optimum washing and elution solvents are 25 mM sodium phosphate (pH 7.0-7.5) and 0.3 M HCl in 60% acetonitrile, respectively. Using the developed method the SA recoveries were found 69-80% calculated from the whole purification process. In particular, in the second 10 mL elution, 70 µg SA in 1 g resin yielded 65% SA recovery, which provided a well-detectable amount of SA with a relatively low level of impurities as analyzed by 400 MHz 1H-NMR. [ABSTRACT FROM AUTHOR]

Monomethoxypoly(ethylene glycol) 5000 (mPEG5K) and 2000 (mPEG2K) were activated to produce mPEG-aldehyde with content over 80%. Effects of reaction time, pH and initial molar ratio of mPEG-aldehyde to insulin on synthesis of PEGylated insulin were studied, taking mPEG5K-aldehyde as a model. On the optimized condition, approximately 90% of product was mono-PEGylated insulin. Mono-PEGylated insulin was purified by ion-exchange chromatography. The purity of each component was analysed by RP-HPLC and the results showed an efficient purification. Matrix assistant laser desorption ionization time-of-flight mass spectra demonstrated that the modification site of mono-PEGylated insulin was PheB1. The biological activity of all PEGylated insulin was also evaluated. [ABSTRACT FROM AUTHOR]

A purification scheme involving ion exchange chromatography on diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose, followed by fast protein liquid chromatography–gel filtration on Superdex 75, was utilized to isolate a laccase from the fruiting bodies of the mushroom Pleurotus eryngii. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose and unadsorbed on CM-cellulose. The molecular mass of the laccase in sodium dodecyl sulfate–polyacrylamide gel electrophoresis was 34 kDa. The laccase activity was maximal at 70°C and remained high at 80°C. High activity of the laccase was maintained at ph 3 to 5. The activity underwent an abrupt decline at pH 6 and 7 and was indiscernible at ph 8 and 9. The laccase exhibited an inhibitory activity toward HIV-1 reverse transcriptase with an IC50 of 2.2 μM. Its N-terminal sequence was dissimilar to those of laccase isoenzymes previously isolated from cultured mycelia of the same species. [ABSTRACT FROM AUTHOR]

A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced byLactococcus lactissubsp.lactisA164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h-1, resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 m NaCl led to approx.>90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield. [ABSTRACT FROM AUTHOR]

A manganese peroxidase (MnP) from a wood-degrading fungus Trichophyton rubrum LSK-27 was purified to homogeneity by anion-exchange chromatography followed by gel filtration. Molecular mass of the purified enzyme was determined to be about 42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Spectrophotometric analysis of the enzyme revealed one Soret maximum at 407 nm, and two visible peaks at 502 and 644 nm, which are consistent with photometric spectra of other MnPs. Mass spectrometric analysis of the digested protein revealed that it had a very high homology to a unique peroxidase (a hybrid of MnP and lignin peroxidase) from Bjerkandera sp. B33/3. Bjerkandera MnP was able to oxidize veratryl alcohol, whereas T. rubrum LSK-27 MnP could not. T. rubrum LSK-27 MnP had the highest pI of 8.2 among MnPs reported so far. The enzyme was stable at rather high temperatures, and when compared with other MnPs, this MnP was more stable in the presence of high concentrations of H2O2. [Copyright &y& Elsevier]

This work presents a method for purification of lysostaphin, a glycylglycine endopeptidase, from the culture liquid of S. simulans biovar staphylolyticus to homogeneity in a few steps. The method includes ultrafiltration and ion-exchange and hydrophobic chromatographies. The enzyme was isolated in preparative amounts with the yield of 51%. Some physical and chemical properties of the enzyme are described. [ABSTRACT FROM AUTHOR]

Purification of green fluorescent protein from a crude solution has been investigated using a combined system of ion-exchange chromatography and size-exclusion chromatography packed into one column of a continuous annular chromatograph. Performing the chromatography with two different sorbents packed into a single column reduces transfer losses and the hold steps between the two separation steps can be eliminated. Appropriate running conditions for the continuous separation mode were achieved by searching for optimal ones in conventional batch-wise operation. Green fluorescent protein was expressed in Saccharomyces cerevisiae, enzymatically lysed and the extract was used as the feed stock. Superdex 200 prep grade was packed into an annular chromatograph (outer diameter: 15 cm, inner diameter: 14 cm) and on top a 1.5 cm layer of the anion-exchange sorbent Source 30 Q. Green fluorescent protein was enriched by the anion-exchange resin and further purified by the size-exclusion gel. Purity was analyzed by sodium dodecyl sulfonate–polyacrylamide gel electrophoresis (SDS–PAGE), Western blotting, fluorescence intensity, and analytical size-exclusion chromatography. [ABSTRACT FROM AUTHOR]

Herein, we synthesized ferromagnetic BiFeO 3 nanoparticles with sizes of 20–60 nm and rhombohedral distorted perovskite structure without any co-formed impurity phases by sol-gel method. Structural, morphological, magnetic, electronic and textural properties of BiFeO 3 nanoparticles were characterized by using XRD, SEM-EDX, TEM, FTIR, XPS, VSM, UV-DRS, PL and BET techniques. The peroxidase was isolated from the Cress (Lepidium sativum) and purified by using precipitation of ammonium sulfate and chromatographic techniques; gel filtration and ion exchange chromatography which is showed wide range of thermal stability, maximum activity in the acidic environment and close to the body temperature. Afterwards, a novel amperometric biosensor based on Cress peroxidase entrapped on BiFeO 3 nanoparticles modified carbon paste electrode was prepared for the first time to sensitive detection of H 2 O 2. The prepared H 2 O 2 biosensor was operated with the aid of choro-amperometry technique by applying −0.8 V vs Ag/AgCl electrode. The developed biosensor exhibited a linear response towards to H 2 O 2 in the concentration range of 2.0 10−7 to 1.0 10−5 M under the optimal conditions and limit of detection and quantification were estimated as 7.0 10−8 M (2.7 μg L−1) and 2.0 10−7 M (8.7 μg L−1), respectively. The repeatability of the electrode was calculated as 19.2 for 2.0 10−7 M (N = 7) and the accuracy was 81.2% for this concentration. In addition, the H 2 O 2 biosensor exhibited excellent stability up to ten days. Finally, the sensor developed was used for the analysis of H 2 O 2 in milk samples and satisfactory recoveries were obtained under the operating conditions. Image 1 • BiFeO3 was synthesized and characterized by various techniques in detail. • A novel H2O2 biosensor-containing BiFeO3 and Cress peroxidase was developed. • BiFeO3 and Cress peroxidase provide synergistic effect for H2O2 detection. • Biosensor was showed excellent stability up to ten days. • The proposed sensor was successfully applied for H2O2 determination in milk samples. [ABSTRACT FROM AUTHOR]

The reproducibility of ion exchange columns was investigated by rigorously evaluating critical chromatographic parameters as well as the performance of these ion exchange columns under various modes of operation. A detailed set of experiments were carried out to determine equilibrium Steric Mass Action parameters, HETP, column dead volume, total ion capacity, and porosity. In addition, the performance of these columns was examined under isocratic, analytical linear gradient, overloaded linear gradient, frontal, and displacement chromatographic conditions. While differences between the two lots were observed with isocratic experiments, the columns exhibited similar behavior under gradient, frontal, and displacement conditions. Furthermore, simulations obtained with the SMA model were shown to predict column performance under linear and non-linear conditions. [ABSTRACT FROM AUTHOR]

Abstract: In this study, nattokinase was purified from Bacillus subtilis using ion exchange chromatography and immobilized upon polyhydroxybutyrate (PHB) nanoparticles. A novel strain isolated from industrial dairy waste was found to synthesize polyhydroxyalkanoates (PHA) and the strain was identified as Brevibacterium casei SRKP2. PHA granules were extracted from 48h culture and the FT-IR analysis characterized them as PHB, a natural biopolymer from B. casei. Nanoprecipitation by solvent displacement technique was used to synthesize PHB nanoparticles. PHB nanoparticles were characterized using transmission electron microscopy and particle size ranged from 100–125nm. Immobilization of nattokinase upon PHB nanoparticles resulted in a 20% increase in the enzyme activity. Immobilization also contributed to the enhanced stability of the enzyme. Moreover, the activity was completely retained on storage at 4°C for 25days. The method has proven to be highly simple and can be implemented to other enzymes also. [Copyright &y& Elsevier]

Abstract: Ion exchange chromatography, one of the major procedures for protein purification, seldom provides single-step purification due to a lack of specific affinity. In this work, a novel and simple method called “back flush” (i.e., reversing the flow direction of elution relative to that of sample loading) was developed to achieve single-step purification on an ion exchanger. Tips for the conditions and operation by back flush are presented. Our study demonstrates, for the first time, the feasibility and dramatic improvement for protein purification by the back-flush method. [Copyright &y& Elsevier]

This study presented a chemical protocol for purification of calcium and magnesium from a single aliquot of biological and geological samples. The separation protocol was based on a two-step cation exchange chromatography using DGA resin and AG50W-X12 (200–400 mesh) resin. Calcium fraction was first collected using 0.25 mL of DGA resin, and Mg fraction was then collected using 0.5 mL of AG50W-X12 cation exchange resin. For Ca isotope analysis, the effect of acidity and concentration mismatch as well as the matrix effect were rigorously evaluated using the Neptune Plus MC-ICP-MS with a wet-plasma mode. Based on repeated measurements on BHVO-2 and NIST SRM 1400, the long-term reproducibility was evaluated at 0.08‰ (2SD) for δ44/42Ca 915a. The δ26Mg ratios of the analyzed reference materials varied from −1.22‰ to 0.03‰ with the external precision better than 0.11‰ (2SD). The robustness of the protocol has been validated by replicated analyses of biological and geological standards. Calcium and magnesium isotopic ratios of all reference materials measured in this study agreed well with previous data within uncertainties. Therefore, the proposed method can be used for simultaneous determination of Ca and Mg isotopic ratios from a single aliquot of biological and geological samples. Image 1 • Purification of calcium and magnesium from a single aliquot of biological and geological samples. • Mg isotopic ratios of GSH-1, GSV-3, NIST SRM 1400, and NISR SRM 1486 were reported for the first time. • The method reduces the amount of acid used, saves time and is more efficient. [ABSTRACT FROM AUTHOR]